JPS5835124A - Preparation of antibody - Google Patents

Abstract

PURPOSE:To collect the formed human lymphoblastoid interferon antibody, by administering an antigen consisting of a complex of at least one of N-terminated peptides of the human lymphoblastoid interferon and derivatives thereof with a carrier to mammals. CONSTITUTION:At least one of peptides of the formula (R is H, H-Thr-His-Ser- Leu-Gly-Asn-Arg-Arg-Ala or H-Ser-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn- Arg-Arg-Ala, etc.) and derivatives thereof is reacted with a carrier, e.g. horse blood serumal albumin, in the presence of binding reagent, e.g. glyoxal, to give an antigen, which is then administered to mammals, e.g. rabbits or guinea pigs. Blood is collectd form the immunized animals to afford an antibody having improved specificity for the human lymphoblastoid interferon and high potency and sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing a plastoid antibody.

In this book, when amino acids, peptides, carrier groups, active groups, etc. are indicated by abbreviations, IUPAC
, IUBO regulations or common symbols Kl in the field of insect wrinkles!
The following are examples. Also, amino acids etc.
IIL, if the optical isomer contains ToC, one is indicated unless specified in 41.

Lal1: Leucine 11g: Iso 0 Ishishi 7L-Nia 5 Niji G1 Island: Kurta! shi Tlhr: ) leonin H*a: histi jishi Say: t rishi Gay: j rishi Jam a asba 5f shifur r! Niashiiniji Asp: Asuba 5f acid Fve Nibu 0 Rishi Z: Kashibobeshi 9 Naka Shi group S 凰: Koheri acid i! Do group 7'##: p-)ruIsrunenyl group Bee:
Interferon Co., Ltd. is an antiviral glycoprotein or protein that is produced when living cells are infected with a virus, and its use can eliminate viruses! It is said that it is possible to prevent or treat disease O, and it has been attracting attention in recent years. Seven things have been revealed in II.

Interfee 0shi is 11 isitafe0shi (Lg*ee
eytmz Istmvfmrem, Lyyip
ho HgztaidInterferes ),!
Lid Ishita Hue O> (FihrmhLmztl*tg
rfmres) and r'fli > 9-7 Eng 0
However, no technique has been developed to purify it into a single glycoprotein or protein K.

The present invention has seven Oa type
To provide a new antibody that provides a technology for isolating and purifying a steroidal antibody, to provide an O antigen and a suitable antibody for the production of the antibody, and to provide a technology for obtaining these. %O for the purpose.

The present inventors have conducted intensive research for the above purpose, and as a result, the N-terminal betutide and its derivatives of a specific human trimester synthesized by the present inventors have been discovered. When used, a novel human primordial steroid interfering antigen can be produced, and by utilizing the wrinkle antigen, a new antibody with specificity for the human primordial lastoid interfering agent can be produced. We have discovered that by using mosquito antibodies in affinity, it is possible to purify the desired human g@interferon. The present invention has been completed based on the above-mentioned new knowledge, and according to the present invention, it is possible to make a trim 5-stoid interface by a simple operation from a synthetic pipe that can be manufactured in bulk and in large quantities. Antibodies that exhibit specific reactivity and are useful for the purification of interferons can be produced industrially and advantageously, thus establishing a technology for purifying human plasma interferon.

The new synthetic betutide according to the present invention has the following general formula (1).
It is expressed as

R-Law-11g -Lea-LeIl&-jZa-
GJs -OH11] [In the formula, R is a hydrogen atom, H-Tkr
-Hip-Ear -Lgs! -(Ay-AznwAr
l -Arc-Arm group, H-5ar-Asp -1n
凰-Prm-GJs-Thr-Hip-5ty-Law
-GLy-Azn -Arc -Arg-Arm group, or H-Tyr-5ay -lap-Laws-Pro
-1n-7"AIP-His-5ay-Lag-Gl
y-Azn-Ar1-Arg -jJ- group. ] or its alligator conductor corresponding to the N-terminal peptide chain. These methods are commonly used for peptide synthesis, specifically, “The peptides.
) J No. 1 (1966) (SckrMdar@n
d Written by ZK Todoroki #, Acadmymie
Press, News York.

U, S, A, 'J or "peptide synthesis" [written by Azumaya et al.

According to the electrical application method described in Maruzen Co., Ltd. Kinsha (19754f), for example, the azide method, the acid anhydride method, the mixed acid anhydride method, the DCC method, the activated ester method ester method, N-hydrochlorinated acid ester method, cyanomethyl ester method, etc.), method using Uridoward reagent I, carboxy! :Taryl method, redox method, DCC/additive (WOMB, HO
Bt % HO 5 m ) method etc. In the above method, either the in-phase synthesis method or the wave-phase synthesis method is used.
Although applicable, wave phase synthesis is preferred.

Usually, general formula (1)
It is produced by the so-called Stellarwise method in which amino acids are condensed one by one, or by a method in which the amino acids are divided into several fragments and subjected to carapuri sifting. More specifically, the above compound corresponds to a raw material having a reactive carboxylic acid group of 10,000 Km of two types of frucmesite divided into two at any arbitrary position of the bond, and the other fraction. It can be produced by condensing raw materials having a reactive amino group such as (1) by a conventional method for synthesizing betatide, and when the resulting condensate has a protecting group, removing the protecting group by a conventional method. Furthermore, when aspartic acid is used in the reaction process to produce a peptide for the general formula, it is often desirable to protect it, and in the final step, at least one of the constituent amino acid residues of betutide is usually protected. All protecting groups are removed from the protected peptide.

In addition, in the synthesis reaction step of bezutide of the above general formula (1),
Functional groups that should not participate in the reaction are protected by conventional protecting groups, and after the reaction is completed, the protecting groups are removed. Furthermore, the functional groups involved in the reaction are usually activated. Each of these reaction methods is well known, and the reagents and the like used therein can be appropriately selected from well-known methods.

As a protecting group for an amino group, for example, carbobenzyl,
tart-butyl-dicarbonyl, tart-7milo+dicarbonyl, isobornyl-deca-dicarbonyl,
P-methocybenzyl-o-deca-dicarbonyl, 2-ri0 rubesidylo+dicarbonyl, dicarbonyl in atamane, trifluoroacetyl, phthalyl, formyl, 0
-Nido-0 phenylsulfenyl, diphenyl-sulfinothioyl and the like. Examples of protective groups for carboxyl groups include alkyl esters (e.g. methyl, ethyl,
ester groups such as divinyl, butyl, tart-dimethyl, etc.), benzyl esters, P-bitobenzyl esters,
Examples thereof include P-methoxybenzyl ester, P-dioxybenzyl ester, becyzyl dibenzyl ester, carbobesidi+shishidragodo, tart-dimethylhydrazide, and tritylhydrazine.

Examples of the guanidino group of alfine include nitro, tosyl, P-methoxysulfonyl, carbovecilyl+cy, isopornylo-dicarbonyl, atamantyl-dicarbonyl, and the like. The quanidino group may also be protected in the form of a salt of a suitable acid such as becisesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid, and the like.

The hydroxyl group of threonide can be protected, for example, by esterification or etherification, but it is not necessary to protect it; suitable groups for this esterification include, for example, acetyl, lower alkanoyl, 70yl, such as benzoyl, penlyyl + dicarbonyl, etc. , elaluo+dicarbonyl, and other groups derived from O carbonic acid. Also suitable groups for etherification include benzyl, tet
These include tart-butyl, tart-butyl, and the like.

Activated carboxyl groups include, for example, the corresponding acids, acid anhydrides or mixed acid anhydrides, azides, active esters (methyl alcohol, ethyl alcohol, benzyl alcohol, bentaf phenol, P
- di-O phenol, N-shidoro + shisakushinyedo,
Examples thereof include N-sodium triaryl, N-sodium triaryl, ester of N-sodium di-5-norbornene:J-2,3-dicarboxylic diimide, and the like. In addition, the peptide bond formation reaction is performed using a condensing agent such as dicyc0+dylcarbosiimide, carboxy! Tarir JlO Karbosi! (In some cases, it may be possible to carry out this procedure.)

Depending on the type of the group represented by the above general formula (L) and the group represented by R, more specifically (I) ~
Manufactured according to method (5).

(I) When R represents a hydrogen atom, A-ALa-B (!1 ↓ H-GA path -OH + 31 A-Ale-GLn-0H (4) ↓ H-ALa-GLn -OH (61 ↓ A-Lg path) -B(61 A-Law-ALa-Gl road-OH(t)HL-attack-
ALg-01%-OH (81↓ A-Lm Ales-B custom) A-Lag-Lawn-7tg- to Zs-OH (9)↓ H-LtII-Lag-Ales-Ghrb-OH switch↓
A -I Z e -B (u)A-ILg-L-
1-L-Law-ALg-GLn-OH↓ H-ILg-Law-Lax-ALa-Gjs-OHf
J field ↓ A-Lm connection-B (II A-Leg-11g-Law-Lag-ALa-Gls
-OH(14↓ H-Lawn-fee-4gs-Leg-Ala-GL
n-OHQ@(II) R is H-Thr-His-5
gr-Law-GLy-Atn-Artl-Arc-j
! In case of +a group, see A-Ar1-B・→ ↓ H-jLg-B first no Arg-Ale -B Exposure H-Shig-AL@-BH C ↓ A-Azn-E@ ■ A-Ass- Arg-Arg-ALa-B 藝41
Lag-ALa-GZs-ON @ ↓ H-AIf&-Arf-Arl-ALa-1,4%-7
16-4, #-LgIl-ALtx-GLm-ORa
ll↓ -C -ON (b) ↓ OHm However, C is based on the following process.

A-Lag-B (II) ↓ H-(Ay-B @ A-Las-GLy-B 114 ↓ H-L-Attack-Gz2B -日↓ , j-5ty-BX A-5ay-Lua-GLy -j - ↓ H-5ar-Las-GLy-B C!14↓ j-
Thy-Hip-B @@ A-'rhy-His-5ay-Law-GLy-B
(I4”alo x is H-5sr-!zp-Law-
Pre-GZ Island-The-Hip-5ay-Lag-G
When indicating Ly-Ass-Arg-Arg-ALa group, 4-pre -E C10) ↓ H-GLn-B@I A-Pro -GLts-B f@ ↓ H-Pro -GLn-B IA ↓ A- 1, mu-B(・) A-LaI&-PrO-GLn-B @fJ↓ H-La5-PrO-GZs-B @2↓ A-AIP
-E U A-Asp-Law-Pra -(Js-B 144↓ A-Asp-Lgs-Pre-GLn-B U↓ II-Asp-Law-Prts -Gts-B @a
↓ l-5ar-B (Foundation) A-5ay -Asp-Law-Pro -Gin-B
1ηH-7hr-Hap-5ay-1,a-GLy
-AssLaw-Ajg-GLs-OH@ A-5-r-Asp-Law, -Pre-Gjs-Th
r-1ha -5ar-4g irises-GLy-Aa buty-Ar
g-Ag-ALa-Lag-ILm-Law-Lava
-Am-GLn-OHw4↓ H-5ay-Asp-Law-Pro -Gjs-Th
r-His-av) R is H-Try-5ay-Asp
-Lag-Pro -GZs -? “Ar-Hlp-5
ay-Lal&-GLy-Ash-Arc-Arc-
In the case of ALa group, A-5ay-Asp-Law, -Pre- to 21m-
Big? )↓ H-5ar-lap-Lag-Pro-Gin-B
-↓ A-Tyr-B Shuchin A-”ryr-5ay-Asp-Las-Pro-GZ
s -B I1, am-jLa-(d*-OH@A-Tyr-5ay -Asp-Law-Pro-GZ
s-Thr --Law-ILg-Lets-Law-
Alga-Gjn-OHM↓ -Lg Juku-Ha-Law-Lag-ALa-GZs-O
H@ [In the above methods CI) to (5), A is a protecting group for an amino group, B is an active group for a hydroxyl group or a carboxylic group, and C
is a guanidino group protecting group of arginine and D is Asva 5e
The protecting groups for F acid are shown below. ] In the above, A is preferably Bat, Z.

The active group of the carboxylic group shown in B is preferably an active ester such as N-methoxydicarbonyl, or a mixed acid anhydride residue such as isobutylic dicarbonyl. , Azide et al., C
Examples of preferable examples of D include nido 0, tosyl, etc., and preferable examples of D include benzyl oxy group and the like.

Above method (I) /Ilf Rito Amino Acid+
The reaction with 31 can be carried out in the presence of solvent O.

As a solvent, eg anhydrous or hydrous dimethyl of each IIO% known to be usable in peptide condensation reactions. Do, dimethyl sulfate, pyridine, dichloromethane, dichloromethane, ethyl acetate, N-methylbiridone, samethyl phosphate tria! or a mixed solvent thereof may be used. The ratio of amino acid (S) and amino acid (2) to be used is not particularly limited and can be appropriately selected within a wide range, but usually the latter is used in an equivalent amount to 5 times the amount of the former, preferably in an amount of ~ The zero reactivity, which is preferably used in 1.5 times the amount, is within the range known to be usable in peptide bond forming reactions, usually about -40 to about 60°C, preferably about -2
0~40℃0Iili! ! Appropriately selected from. The reaction time is generally about several minutes to 30 hours.

In method (I), the reaction between peptide (5) and amino acid (6), the reaction between peptide (ta) and amino acid (6), the reaction between peptide and amino acid (11), and the reaction between peptide and amino acid (11), The reaction between Tido and the amino acid (Gl) can be carried out in the same manner as the reaction between the amino acid (!) and the amino acid (3) described above.

In addition, the amino acid Orj and the amino acid in method (II)
Reaction with η, reaction between Peptide Kenshi and Amino Acids, reaction between Bezutide Akane and Amino Acids, Pezutide 6! ! Rumors and the reaction between Bezuchido and Bezuchido, the reaction between Bezuchido and Bezuchido,
Reaction between amino acid (6) and amino acid 4, bezutide CIO
The reaction between and amino acid (goods) and the reaction between bezuyodo (goods) and bezuyodo μs to 4 ptiw! l! It can be done. Furthermore, in the method @), the reaction between amino acids and amino acids, the reaction between Pezuyodo and amino acids, the reaction between Pezuyodo and amino acids, the reaction between peptides and amino acids, and The reaction between Bezutide supplement and Bezuyodo (foundation), the reaction between Bezudo and amino acid 11) in method (5), and the reaction between Bezuodo and Bezuyodo (2) are also the same as above. You can do it in the same way.

Bezutide (4), (7), (
9), 01, H1 ■, -1 (2), (goods), -1 glance,
The elimination reaction of the protecting group A of ramen, taku, ku, i4η, ichi and - is carried out by a conventional method. Such methods include, for example, reductive methods (e.g. hydrogenation using palladium, vanadium kurogo catalysts, reduction with metallic sodium in liquid acetic acid), acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, methane). Examples include acidolysis with strong acids such as sulfonic acid and hydrobromic acid.

Hydrogenation using the above catalyst can be carried out, for example, at a water bulk pressure of 1 atm and 0
It can be carried out at ~40°C. The amount of catalyst used in your company is usually about 100 wI to about 11 wI, and the reaction is generally completed in about 1 to 48 hours. In addition, the above acidolysis was performed without a solvent.
The reaction is usually carried out at a temperature of about 0 to 30°C, preferably 0 to 20°C, and the reaction is generally completed in about 15 minutes to 1 hour. The amount of acid to be used is usually about 5 to 10 times the amount of the raw material compound. In addition, in the reduction with metallic sodium in liquid asimonia, metallic sodium is used in such an amount that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes. This reduction is usually -40--70
It appears at about ℃.

Furthermore, the protecting group CC) of Bezuyodo (2) and the protecting group (D) of Bezuyodo can be similarly disengaged by the above-mentioned reductive method.

Amino acid (2) used in the above methods (I) to GV)K
, (6), C11), rJ Fu, Kan, (foundation), -1- and If) may be known commercially available products, and peptide reference F4, Betsu, 輛, (t), -1←η, and For (conversion), known commercial products or those obtained by a mixed acid anhydride method, an azidation method, etc. can be used. The mixed acid anhydride method is carried out using carboxylic acid in the presence of a basic compound in a suitable solvent. Examples of the alkali-halocarboxylic acids that can be used include methyl formate, methyl formate, ethyl formate, ethyl formate, isobutyl formate, and the like. Examples of basic compounds include triethyla! ! Niji, trimethyla! cy, pyridine, dimethylanilisi, N-methyl heptylanilysi, 1.5-,; azabishikuo (4,3,0)] nonene-5CDEN',
1,5-diazabisifu o [5,4,0) undecene-
5 (DBU), i, 4-,; Azabishik 0 (2, 2
, 2) Organic bases such as octane (DAECO), and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The solvents to be used include various solvents commonly used in the mixed acid anhydride method, specifically, halogenated hydrocarbons such as methylene chloride, RI00 form, and dikuoo etashi, and aromatic hydrocarbons such as benzene, toluene, and silane. etc., diethyl ether, Tet-5 and Do-O-5
Ethers such as N, dimeth + ester, esters such as methyl acetate and ethyl acetate, N,N-dimethane, etc. Do, dimethyl sul in water, he + samethyl lysic acid thoria! Examples include polar polar solvents such as d, d, d, etc. The reaction is carried out at -20 DEG to 100 DEG C., preferably at -20 DEG to 50 DEG C., and the reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours.

In addition, in the azidation method, first the activated carboxyl group,
For example, this is carried out by reacting dorazine hydrate with a carboxyl group activated with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol in an appropriate solvent.

Examples of the solvent that can be used include dioxane, dimethylformanide, dimethylformanide, and a mixed solvent thereof. When using Shidorajishi hydrate, the amount of activated carboxylic group is usually 5 to 20.
The amount is preferably 5 to 10 times the temperature. The reaction is usually carried out at a temperature of 50°C or lower, preferably -20 to 30°C.
It will be held at In this way, a compound (hydrazine derivative) in which the carbo+syl group of the terminal amino acid is substituted with hydra, ;cy can be produced. A compound in which the carboxyl group of the terminal amino acid is substituted with azide is produced by reacting an acid conductor with a nitrite compound. Hydrochloric acid is usually used as the acid. Examples of solvents that can be used include hydroxide, dimethylformanide, dimethylformanide, and mixed solvents thereof. Further, as the nitrite compound, for example, sodium nitrite, isoamyl nitrite, nitrite, etc. can be used. Such a nitrite compound is usually used in an amount of 1 to 2 times the amount of dorazine, preferably 1 to 1.5 times the amount of nitrous acid. The glare reaction is usually -20 to 0°C, preferably -20 to -10°C.
The reaction is generally completed in about 5 to 10 minutes.

The bezutide of general formula (1) produced as described above can be obtained by means of separating the peptide from the reaction mixture, such as extraction, distribution, etc.
It is isolated and purified by the following methods.

In this way, a synthetic peptide represented by the general formula (1), that is, a tricyphod-5stoid-interfered 0-cyon-terminated peptide and derivatives thereof are obtained.

The synthetic peptide thus obtained is 1. 1
By introducing radioactive iodine such as (e.g., radioactive iodine), it can be used as a labeled beutide, which is a raw material for the production of labeled antigens used in Radioim's 7-day method (RIA method). The above radioactive iodine can be introduced using the usual iodination method, such as Ri0! Oxidative 3-dation method using T [Fl', M,
Hunter antiF, C, Greenwood
; Nature, 194, P495 (19
62), Biocihmwh, J., 89, P.
114 (1963)]. For example, the above-mentioned iodination method can be carried out using a suitable solvent such as 0.2 M phosphate buffer (pH=7.4). : It is carried out for about 10 to 30 seconds at a mild temperature in the presence of N.T. Bezutide, radioactive iodine and radioactive iodine used! For example, when one radioactive iodine is introduced into TiO-Shishi, the ratio of radioactive iodine to be used is approximately 1 mm + 1 L for each nanomole of Tyr-53 molecules contained in Bezutide. It is best to use about 10 to 100 nanomoles of 0-RaeshiT, and when introducing two radioactive iodines into Ti0-Shishi, add radioactive iodine to 1 nanomol of Tyro-93 molecules contained in Bezutide. About 2mm Yuri, Ri05
It is preferable to use three types of T at a temperature of 10 to 100 nm. The radioactive iodine-labeled betatide produced in this manner is isolated and purified by conventional separation means such as extraction, partitioning, chromatography, dialysis, and the like.

The product thus obtained can be freeze-dried and stored if necessary.

Furthermore, the synthetic peptide represented by the above general formula (1) can be used as a hatching material for the production of human plastidoid antigen.

The method for producing the human plastidoid protein antigen described above will be described in detail below.

The present protein interferon antigen contains at least one synthetic compound represented by the above general formula fl).
It is produced by using a hapten as a seed and reacting it with a carrier in the presence of a carrier-binding reagent.

In the above method, the carrier to be bonded to the adhesive includes:
High-molecular natural or synthetic proteins commonly used in the production of antigens can be widely used. Examples of the carrier include animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin! Animal sera such as rainbows, horse serum dioplasts, bovine serum pox, rabbit serum pox, human serum I5, sheep serum ppurine, horse chi pox, etc. , cattle chioori0zurin, lip 4 yori 0ri0purishi, human food 0ri0burishi, shitsujichi 0ri0zurin, etc., animal chioori 0zurin, horse 7ri0bin, 0 pudding for cows, 0 pudding for rabbits, 0 pudding for humans,
Proteins extracted from animal products such as sheep and purines, and proteins extracted from animal species and roundworms (Ascaris extract, !l!l see 1987-16414) ), polyglutamic acid, lysine-curtamic acid copolymer, and copolymer containing ornithine.

The Ascaris extract is manufactured by O. Ascaris Extraction Co., Ltd., as detailed below.
It is extracted from the pulverized product of 1) according to a conventional protein extraction method.

As the extraction medium, various known protein extraction solvents such as water, physiological saline, 50% methanol or ethanol aqueous solution, and near-neutral buffer solutions can be used, with physiological saline being particularly preferred. More specifically, the above extract is manufactured by a company, for example, as follows. That is, after removing the contents of the porcine roundworm and washing it with physiological saline, it is preferably shredded to facilitate extraction, and the shredded material is added to a protein extraction solvent such as physiological saline and homogenized. while extracting.

This extraction is usually carried out at low temperatures, preferably at about 2 DEG to 10 DEG C.

The extract obtained in this manner is then centrifuged, the supernatant is collected, and the dialyzed receding liquid is freeze-dried, or the dialysate is centrifuged again, the supernatant is collected, and the supernatant is removed, followed by freeze-drying. In this way, the desired Ascaris extract is produced. This can be further utilized in the present invention, if necessary, by conventional protein purification methods such as dialysis, gel filtration, adsorption, and chromatograph purification.

Further, the above-mentioned Ascaris extract is, for example, J, fwh@s
m.

111.260-268 (197!S), J, 1w
nwnthm,.

122.302-308 (1979), J. Imm.
gm,, 98°893-900 (1967) and Al
l1. J, PhyziaL,.

199.575-578 (1960) ft.
Turtle or further purified products thereof may also be used.

As the hapten-carrier binding reagent, a wide range of reagents commonly used in the preparation of antigens can be used, and specifically, those that cross-link amino groups, such as cryo+sal, Ma0:J dialdehyde, Aliphatic dialdehydes such as geltaraldehyde, succialdide, and adipaldehyde, which crosslink thiol groups with each other, such as N,N'-e-phenylyl-maldehyde, x,
y'-Shimaleinide compounds such as phenylemaleimide, cross-linking amino groups and thiol groups, such as metamaleimide besylyyl N-human 0+ciskushi! doester, 4-(malei:domethyl)-Schif0 to 1-carbo+silu N'-citro+cissuccinimide ester, maleimidocarbo+silu N-human 0+cissuccinimide ester compound, amino group and sil in the carbo Reagents used in the usual -butide bond formation reaction to form an anide bond between groups, such as N,N-disyc0 to +
Zylcarbocyinide, N-ethyl-N'-dimethylaminocarbodiimide, l-ethyl-3-diisopyraminocarbodiimide, 1-Schiff0, Nakasil-3-(2-
Examples include dehydration condensation agents of carbodiimides such as 1: luforinyl-4-carbodiimide,
Furthermore, it is also possible to use a combination of a 5-alinium allylcarbo 2/M such as Fip-t allinium phenylacetic acid and a conventional peptide bond-forming reaction reagent, such as the above-mentioned dehydration condensation agent.

The reaction for producing the antigen of the present invention can be carried out, for example, in an aqueous solution or at a pH of 7.
~toO The reaction is carried out in a normal buffer solution, preferably in a laxative solution having a pH of 8 to 9, at o-40°C, preferably around room temperature. The reaction Fi is usually completed in about 1 to 24 hours, preferably 3 to 5 hours. Examples of typical buffers used above include the following.

0.2N sodium hydroxide-0.2M boric acid-〇,2I
Potassium chloride buffer, 0.2 M sodium carbonate') Mu-0,2j/J U1l
-0.2M potassium chloride weak solution, 0.05M sodium tetraborate -0.2M boric acid -0,
05M sodium chloride buffer solution, 0.11 potassium dihydrogen phosphate-0.05M sodium tetraborate solution In the above, the proportions of the hapten, the hapten-carrier binding reagent, and the carrier can be determined as appropriate; It is preferable to use the carrier in an amount of 2 to 6 times the weight, preferably 3 to 5 times the weight, and the hydrogen-carrier binding reagent to be used in an amount of about 5 to 10 times the weight. Through the above reaction, a human plastidoid antigen consisting of a beta-carrier complex in which a carrier and a hapten are bound is obtained through the mediation of a hapten-carrier binding reagent.

Antigen radiation obtained after the reaction is completed according to the usual method, for example, dialysis method,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc. Furthermore, the axillary antigen can be preserved by the usual freeze-drying method.

Thus, a plastoid interferon antigen consisting of a complex of the N-terminal heptide of the plastoid interferon represented by the general formula 11) and its derivatives with at least a 41s carrier is obtained. . The antigen usually has an average ratio of 5 to 20 molecules per protein i'e.
All of the t-linked antibodies make it possible to produce highly specific antibodies against human lastoid interfaces with good reproducibility. In particular, the binding ratio of betutide to the above protein is 1:8 to 15.
It is preferable that antibodies with higher oFi, higher % isomerism, higher titer, and higher sensitivity can be produced.

Preparation of antibodies using the antigen obtained above is carried out as follows. That is, it is carried out by poisoning a mammal with the above-mentioned antigen and collecting antibodies produced in the body.

There are no particular restrictions on the mammal used for antibody production, but it is usually desirable to use rabbits or rats. When preparing antibodies, dilute the antigen obtained above to an appropriate concentration with physiological saline, mix it with Freund's auxiliary solution, and suspend it. The solution can be prepared and administered to the incubator. For example, rabbits may be injected with the above suspension (antigen amount: 0.5-511 P/dose), and then administered 2 to 5 times every 2 weeks. It is sufficient to immunize by administering for 10 months, preferably 4 to 6 months.The antibody is collected after the final administration of the suspension, when a large amount of antibodies are produced, and after 1 to 2 weeks of energization after the final administration. In particular, according to the method of the present invention, based on the characteristics of the anti-JI [OIF#] used, the human serum protein is It has an advantage of having extremely excellent specificity for 5-stoid interference, and of being able to obtain antibodies with high titer and high sensitivity.

The thus-obtained 5-stoid interferon antibody has particularly excellent specificity for the above-mentioned 9-hydrin plastoid interferon. I)
, Triumph 5 using the RIA method, which is desired in this field.
It is a useful product that enables the quantitative determination of steroid interferon with high accuracy.6. Furthermore, the antibody can be used in enzyme immunoassay (EI) and fluorescence immunoassay (FIA) methods by labeling the antibody with an enzyme or a fluorescent substance. Furthermore, the antibody is a known O
It can also be made into an insolubilized antibody by reacting with a substance to be insolubilized.

In order to explain the present invention in more detail, examples of the present invention, an antigen using the same, and production of antibody Q from the antigen will be given below, but the present invention is not limited thereto.

In addition, RffM in each production example is 0 for the thin layer on silica gel.
It was measured using the following mixing method on Matoku 5 Fee.・Rf...l-butanol-acetic acid-water (4:1:5)R
f...1-butanol-pyridyacetic acid-water (15:
10:3:12) <Synthesis of peptide> Production example 1 1. Z-ALg-GJs-OHO production Z-ALg-
05I14-80 f Onato 5 Human 0 Fu 5 White 〇-solution, H-GZs-OK 2. 40 ml of water solution of 191 and triethylamine 2. Add lO- and stir at room temperature for 20 hours. Hydrofuran and water are distilled off, and the residual liquid is extracted with iso-butanol. The extract was washed with dithiacetic acid, and the Ztanol was distilled off. The precipitated substance P was reprecipitated with methanol-ethyl acetate to give Z-ALg-GJs.
-ON' Get -$871.

Rf, 0.41 Rf, 0.56 Elemental analysis value (C engineering 6'2 engineering N, 06) Calculated value ('
II) 854.69 B6.02 ff11.
96 Actual Value (*) C54,5Of6.51 #1
1.622(a), Production of H-ALa-Glt>-OB Z-jLtx-GL-OH 3,50t in water 50m
and methanol (30 mK) and catalytic reduction using palladium as a catalyst to obtain H-A Mu G Mu 011.

R7, (LO4 2(Jl), 2-LeI&-ALa-GLh-OH
O production Z-L-1-O5t & 3.97t, H-Alg-GZn-011 obtained in (α) above and triethylamine 1.39Mt-dimethyl are dissolved in 50m7 of this Lumuaed and stirred at room temperature for 20 hours. Dimethyl car rumua! The residue was extracted with ethyl acetate. The extract is washed three times with IN-quesiic acid and five times with water. Ethyl acetate was distilled off, ether was added to the residual liquid, the precipitate was removed and dried, and reprecipitated with methanol-ethyl acetate.
-Law-ALa-Gls-OK 2.15 f is obtained.

Rj, 0.49 Rf, 0.62 Elemental analysis value (as C22N, □407) Calculated value C1)
056.88M6.94 N12.06 Actual value (
1ri C56,41N6. BO#12.183(g)
, H-Lag -ALa-GZs -OHf) Production Z-Lg*-Δ1m -Glm -ON 2.1 Of
Add 20ml of acetic acid solution containing 25thihydrobromide to
Leave it for a while. Add dry ether to the reaction solution and
Obtain ewb-Alg-GLh-01.

Rf": 0.10 3(b), H=Z#m-Law-jjPg-Gl
Manufacturing of m-OB Z-Lg@-05m 1.96 f,
) ethylamine 0.63d and H-Law-Al
g-GLh -OH in dimethyl lumatrido 50111
Dissolve in 7! ! Stir with a turtle for 20 hours.

Dimethylformamide is distilled off, IM quesic acid is added to the residual liquid, and the precipitated crystals are taken off, washed with water until the p liquid becomes neutral, and dried. Wash with methanol-ethyl acetate and add La-0 to Z-Lat&-LaI&-AleL-.
H1,63t is obtained.

Rf, 0.58 Rf": 0.64 Elemental analysis value (as C26H,, N, 08) total j[('
J) C5B, 22 N7.50 N12.12
Dormitory measurement (%) C57,85N7.90 #11.
964(a), H-LaIl-LeK-Ala-G
js -ON (D production Z-Lag-Law-jZg-
Add 25-hydrogen bromide-containing vinegar 1[liquid 2o-] to GE Island-OH1, 5Of, stir at room temperature for 1 hour, add dry ether to the reaction solution, remove the precipitated solid, and add H-Law.
-Law -ALa -Gln -OH is obtained.

R7,0,19 4(A), Z-ILg -Lag-LgAla
-GLn-OHtl) Production Z-ILa-O5IIL1-
41 f, obtained above, 9H-LalL-Lag-AL
Dissolve a-GL*-OH and triethyl amine 1O-36d in dimethyl fluoride 50- and stir at room temperature for 20 hours.Dimethylformamide is distilled off and the residue is
Add N citric acid, collect the precipitated crystals and wash with hot methanol to obtain Z-ILa-Lam-LaK-ALtx.
-GZs -ON 1.17 f t” 2).

Rf, 0.63 ff/, 0.71 Principal Analysis"(C34'54N6'9 ToL Te) Calculated Value (IG) ('59.11 N7.87 N1
2.16 real chaotic fiic%) C59,23N7. B.O.
N12.025(a), H-11g -La
g -jZg-Gln-OH (1) production Z-11a -
Law-Leu-Ala-Gin-OH1,10f K
Add 15 M of acetic acid containing 25 thihydrobromide and stir at room temperature for 1 hour. Dry ether was added to the reaction solution, and the precipitated solid was collected by V-ILg -Law-Lau-ALa -
Obtain GZ Gar U.

Rf, 0.25 5(b), Z-Law-ILa-Lgu-Lau-A
la-Gin-OHlDH Z-Law-05% O-69fsH obtained above
-11g-Lg, mu -11a-01%-OH and 22 ml of methylformamide were dissolved in 50ml of methylformamide and stirred at room temperature for 20 hours. Dimethylformanide is distilled off, IN succinic acid is added to the residue, and the precipitated substance is collected in an oven, washed with water and dried until the F solution becomes neutral. Wash with hot methanol and prepare Z-Lag-B.
g-Lm Dai-Law-jt@-GmuOH1,10ff
tf4:b.

Rf”: 0.5B Rf, 0.71 Elemental analysis value (Ca. '65 N7') Calculated value (11) C59,75H8,15N12.19 Actual value (-) C59,60H8,02#11 .926,
H-LaI&-11a -Lam-Law-Ale-G
is-QHOH construction Z-1℃ connection-ILa -Leg-La
g -, #a-(da-DH0,5Of methanol 5
H-Lag-ILa-La@-
Law-ju@-Glm-OHfib. Below this is “
It is called peptide A.

Rf”: 0.23 Rf, 0.6 elemental analysis value (C32I5.N, 0.-2 H20 and shite) calculated value (IG) C54,45H8,99N 13
．． 89 Actual scooping value (%) C54,50H8,81N 1
3.98 Production example 2 1. Boc-ALa-NHNH2O production Ihc
-jLtx-OH4,991%m2-NH-24,36
If W: I! Do5.44
f was dissolved in tetrahydrofone 150- and heated at 4°C for 20
Stir for an hour. The precipitated solid was removed by distillation, and the F solution was distilled off.
Add ether to remove precipitate V, reprecipitate from ether and petroleum ether to obtain Bac -Ala -NHNHz
? , O: $ eλ/ to obtain f/, 0.79 fff, 0.81 Elemental analysis value (C,, I2.N, 05T1.TE) Calculated value (91) C56,96H6,8'I N 12.
45 fruit 111 [(1g) ('56.81 H6,
4971'12.342(a), H-ALa-NH
NHZ production Boc-ALa -NHNHz 3-00
f was dissolved in trifluoroacetic acid lO-, left at room temperature for 15 minutes, and then the trifluoroacetic acid was distilled off and dried to form H-AL@
-NHNH2 is obtained.

Rf, 0.51 2(b), Bat-Ary(No2)-ALa-N
Production of HNH2 Btsc-Arg (H02)-OH2
, 84F was dissolved in a solution of 40 aj of sodium chloride, 40 aj of N-methyl, and 4 bottles of N-methyl, and after cooling to -15°C, 1900 isobutylene and 1.17 m of lumate were added.
Stir occasionally for a few seconds. This IcH-noL a-NHNH
Add the dimethyl lumuaed 211411 solution and triethyl amyl 1.24 solution of z, and stir for 1 minute. 0℃
The mixture was heated at 40° C. for 5 minutes, then at 40° C. for 2 minutes, and then stirred at 40° C. for 15 minutes. After distilling off Na5-hydrofuran and dimethylformamide, the mixture is extracted with ethyl acetate. The extract was washed with IN citric acid, followed by saturated sodium bicarbonate, and then saturated brine, ethyl acetate was distilled off, and reprecipitated with ethyl acetate-ether to give Bee-Ag(NO2)-Ala-N.
Obtain HNH2!3.70 f.

Rf”: 0.68 Rf, 0.79 Elemental analysis value (C22, y, o8) Calculated value (-
) C49,06N6.36 N20.80 Actual value (-) C49,40#6.72 N20.43
5(a), H-,4r! (AI'Oa) -Arm
-NHIIH2O@Zo Bee -Arc (NO2)
-ALa-NHNHz 3.67 f was dissolved in 15 m of trifluoroacetic acid, left at room temperature for 15 minutes, crystallized by adding dry ether, and the crystals were collected to obtain H-"!I (7'
02) -jja -NHNH2f % le.

Rf, 0.20 3(b), Bac-Arg(NO2)-Arl(N
Production of 02)-ALg-NHNH2Bet-Arl(NO2)-OR2-17f
50ml of human 0 flage and 0.69ml of N-methyl alcohol
11/Dissolve in solution and cool to -15℃
Add 0.89 m of Lumate and stir vigorously for 50 seconds.

To this, nine H-Arg(No21)- obtained in the above (-)
Dimethyl rate of ALa-NHNIIZ: 39m
and triethylamine 0.95-solution and stirred for 1 minute. After warming at 0°C for 5 minutes, then at 40°C for 2 minutes, stir at room temperature for 15 minutes. The tetrahydrofluoride and dimethyl hydroxide are distilled off, and the residue is extracted with ethyl acetate. The extract was washed successively with a mixture of IN-quesiic acid and saturated sodium bicarbonate water, and then ethyl acetate was distilled off. Ihc-ArlC was reprecipitated with acetic acid and ether.
N02) -Art CCN02) -AL -NHNM
Obtain Z'3.70f.

Rf”: 0.58 Rf, 0.75 Elemental analysis value (as C28H, N engineering, 0AE) Calculated value (phantom C45,46N6.13 N24.61 Actual value C1) C45,13ff5.71 N24.51
4(a), H-Arc(NO□)-Arg(NO2)
-1La-NHNHz production Bat-Arc(No2)-Arl(NO2)-ALa
-NHNHz 3.0Of trifluoroacetic acid 201d
After standing at room temperature for 15 minutes, dry ether is added to crystallize.

Take the crystal V and H-Arl (No2)-Arc (
NO2) -ALa-NHNH2 is obtained.

ff/”:0.11 4(A), BaC-Azn-Arl(No2)-Δ
'! Production of I (N02) -AL @ -NHNHz H-Arc (NO2)-Arg (JVO2)-AL
Dissolve a-NHNHz in DMF5Qsc/, add triethylamine 0.56- and Bac-Ass-0885 to it.
Add 2.17f and leave at 1i1 temperature for 20 hours. Dimethyl rate rumua! The residue is extracted with butanol. After washing the extract with 2% acetic acid, ether was added to crystallize it, and the crystals were collected in a furnace and reprecipitated (with methanol-acetic acid) to give 13ec -Arm -Arg (NO2)-Ar.
c (No2) -Ala -NHNHz 2.64
Get ft.

Rf": 0.40! Rf: 0.72 Elemental analysis value ('32H51N, 5'l, Toshite) IHI
EII (li) C45, OI H6, 02N2
4.60 actual mlli (II) C44,80H5
, 85N24.124(c), Boe-Azn-A
Production of g-Arc-Ala-NHNH2Ba c-As
s -Ar1(N02)-Arc(NO2)-Ale
-NHNHz2.5Of methanol 30m and 3o-
Bee-Arg-Arg-Arc-Al was dissolved in a mixture with acetic acid and catalytically reduced using palladium as a catalyst to give NHN.
H22,2Of is obtained.

R/': 0.06 Rf": 0.40 Elemental analysis value (C2,, H, 7N, 0.-2CH, CC
Ap H20) Total jEm (%) C43,80H7,48N23.
71i 11m ('II) C43,51H7,62N
25.455-Z-Law-Gly-()C2HB
Combining the 111N-methyl hexafluoride 1.86 su with Nato 5 human 〇75)6011uK,
-Add OH4-85f. Cool to -15°C, add 2.41 ml of Isoptiluc 00 Nelumate, and stir vigorously for 30 seconds. KotL K H-Gly-QC2H5-
HCl 2.54 t cD dimethyl lumanide 40 resistant solution and triethyl a! m: Add J2.56m, 1
Stir for a minute. After warming at 0°C for 5 minutes, then at 40°C for 2 minutes, stir at room temperature for 15 minutes. Nato 5 human 0 furan and dimethyl book rumua! After distilling off the residue, the KIM quench was added to citric acid, and the precipitated crystals were subjected to PNR. The P solution was washed with water until it became neutral, and the precipitated large crystals were removed with F, dried, and reprecipitated with ethyl acetate-ether to form Z-LgIII&-(dy-
0'C2H54,6By is obtained.

Rf”: 0.80 Rf”: 0.77 Elemental analysis value (as C engineering, H26N205) Calculated value C1
) C61,70H7,48H7,99 Actual side value C%)
C61,51H7,32H7,806(α), H-
L au-GL y -0C7snomanufactureZ"L
g*-GL y-()C:aHs 3-12
f is dissolved in J'))-L50d and IN salt 118.90117, and subjected to catalytic reduction using palladium as a catalyst, H
-Law-GLy- situation" 2H5 is obtained.

Rf, 0.41 6(A), Z-5ay-1ay-GLy-QCL!
Preparation of H5Z-5et-NHNH22,+8f”
Dissolve in 20 iu of JfL*'RtL7E and 4.89 il of 6N hydrochloric acid/dioxide, cool to -15°C, add 1.31 - of isoanyl nitrite, and stir for 5 minutes. Then, triethylamine 4111Lt is added to neutralize.

H −L# s −GL y −O obtained in the above (→
C"ais ・I HCl and triethylanine 1.24
The above reaction solution was added to a 11 J dimethyl solution of Lumuaed 10d, and the mixture was stirred at 4°C for 20 hours.

After distilling off the dimerium amide, the residue was extracted with ethyl acetate, and the extract was washed successively with IN-citric acid and saturated brine, and then dried over magnesium sulfate. After distilling off the ethyl acetate, it was reprecipitated with ethyl acetate to give Z-5ay-Lag-
GLy-QC2M, 2.64 f is obtained.

Rf, 0.78 Rf": 0.85 Elemental analysis value ('21'!! 60H6,88N9.637
(a), H-Ear-Law-GLy-0C2
H5O production Z-5ay-LaaL-GLy-QC2H5
2-501 was dissolved in 10 m of 10 thiacetic acid and 50 m of methanol, and catalytically reduced using palladium as a catalyst to obtain H-5.
61-Laμ-GA y -QC2M is obtained.

Rf”: 0.31 Tu4), Z-Thr-Rig-5ay-Lag-
Production of 01y-QC2H5 Z-Thr-Hip-NHN
H22-54f t'; Methylformamide 20Jl
/and 6N-hydrochloric acid/! Dissolve in 4.19 a/ml of sashimi, cool to -15°C, add 0.84 m of isoamyl nitrite, and stir for 5 minutes. Then triethylamine: J3.
Add 51- to neutralize. Nine H-5a obtained in the above (α)
t-LaI&-GLy-QC2H, and 0.7911d of triethylamine in 201 methylformamide solution,
Add the above reaction solution and stir at 4°C for 20 hours. After distilling off dimethylformamide, the residue was extracted with ditanol,
Wash the extract with water. The solvent was distilled off and recrystallized from methanol-ethyl acetate to give Z-Thr-His-5ay-L.
awn-Gly-QC2H54-311 is obtained.

Rf, 0.55 Rf, 0.71 Elemental analysis value (as C3 engineering H45N, 03/H20) Calculated value (1) C64, OOf8.14 N16.8
5 Actual column value (-) C64,48ff8.10ff
16.548(t), Z-Thr-H*z-5et
-Lava-GZy-NHNH20 production z-rm v-H
is-5ay-Law, -GLy-OC2#5 4.5
Of is dissolved in 20-methanol, 5.18114 human 5-dihydride monohydrate is added, and the mixture is left at room temperature for 20 hours.

Ether is added to the reaction solution, and the precipitated crystals are separated from P and dried. Wash with hot methanol and remove Z-Tfr-His-5.
ay-1pnb-GLy-HEME22.55 f is obtained.

Rf, O,17 Rf, 0.57 Elemental analysis value (as C29H43N909) Calculated value (!
16) C52,64H6,55N19.05 actual value (%) C52,55H6,44N 19.098 (
b), H-Azn-Arc-Ary-ALa-La
x-ALa -Lag -Lms -ALa-GLn-
Production of OH BoC-j#5-Ar! 1-Arg-jet
-NHNH20-78f't-dimethylformamide 8d and 6N-hydrochloric acid/diosushif, Q3Mlic
After dissolving and cooling to -15°C, isoanyl nitrite Q, 35
Add 11 Ll and stir for 5 minutes.

Next, 0.87 m of triethylamine was added to neutralize.

Pezuyodo A, that is, H-Law -ILa -Lax -L
aw -ALa-GLn-OH,) Rie yo Lua! N0.
87117% dimethyl lumamide 20111 and 5-methyl phosphate tria! The above reaction solution was added to the detowash solution, stirred at 4°C for 24 hours, and further mixed with Bee
-Ass -Arg-Arc -ALa-NHNI et al. 0
．． Add 39F converted to azide and stir for 48 w#. Jime Yoruformua! The residue was extracted with ethanol. The extract is washed with water and the butanol is distilled off. Ether is added to the residue to crystallize it, and the precipitated crystals are designated as P*. Wash with water and dry with pentoxide. Obtained 1
3ee-,4pn-Ary-Arg-ALg-Law
-Ije-1pn-Lag-ALa-GLs -OI was dissolved in triple 0-acetic acid 3dK, left at room temperature for 15 minutes, and then dry ether was added to precipitate, which was then dissolved in Fjm
After drying, it was purified with Sephadux G-25 (eluent: 50-acetic acid) to obtain H-Azn-Ar! 1-Arc-Ala-L
ag-Be-Leg-Lea-ALa-Gin-OH1
Obtain 20 capes.

Rf, o, ot Rf, 0.35 Elemental analysis value (C5, H9aN, 80.3-2 CH3C
As O0H・5H2o) Calculated value (chi) C41,95H8,19N18.30
Actual value (11) C47, 66H8, 41#I8.6
2[α), -185,44(C-0,57,1M acetic acid)9, H-Thr-His-5ay-Law-G
ly -Ash-Arg-Ag-ALa-Law-AL
Production of a-Law-Lag-ALa -Gin-OH Z-Thr-Hip-5ay-Lgs-GLy -NH
Dissolved in NH2125''Ft dimethyl fluoride lQm and 6N-hydrochloric acid/;
After cooling to -15°C, add 0.025 d of isoanyl nitrite and stir for 5 minutes. Next, add 0.105 d of isoanyl nitrite.
Add to neutralize. H-,1ax-Ar1-Arg-Al
g-Lag-1ie-Law-Law-ALa-GZ
a-011110111 and triethylanil 0.015
Add the above reaction solution to a solution of 10 m of dimethyl phosphoric acid trianide and 6- of dimethyl phosphoric acid trianide,
Stir at 4°C for 24 hours. Furthermore, Z-Thr-1ha-
5ay-Law-Gly-NHNH2125"Ill was added to 75-d and stirred for 48 hours. ◇Dimethyl rumamide was distilled off and the residue was extracted with tanol. The extract was washed with water. Butanol was distilled off. -IZa
-Law-Law-Alt-Gln-OH is dissolved in methanol 5Qm and 10-acetic acid IQm and catalytically reduced using palladium as a catalyst. The catalyst was removed, followed by methanol distillation, and the resulting residue was purified with Sephadex G-25 (eluent 5091 acetic acid) to obtain H-Thr-Hip.
-5ar-Law-Gly-Ass-Arg -Ar5
1-Ala-Lag-Be-Law-LεμmJZa-
Pull: Obtain 5-0H125 Cape. Hereafter, I will refer to this as “Bezuyo de B”
” is called.

Rf, o, ot Rf, 0.38 Element 5+ Digit value (C72H127N250ao°2 C
As H3CO0H-4H20) Calculated value (*) C49,21H7,77N13.87
Actual measurement value CTo”) C49, 60H7, 92N18.
54 [α] , -66,76CC mouth 0. 2.1M acetic acid) Production Example 3 1(g), Production of H-GLs-NHNHBoc Z-
GL@-NHNHBtsc 7. Soak OOf in methanol 5Qsl, Ba5! catalytic reduction using aluminum as a catalyst to obtain H-01%-NHNHIhc.

Rf, 0.37 1(A), Production of Z-pro-GLm-NIINBβ. After dissolving in gQNl and cooling to -15°C, add Isoptiluc00 Nelumate 2.311j and stir intermittently for 30 seconds. This is K
The above (g)-1” obtained H-GL 5-NHNHB
o e C) ”;Methyl rate Lumua!Ad 30- Add the solution and stir for 1 minute.

Warm at 0°C for 5 minutes, then at 40°C for 2 minutes, then stir at temp for 15 minutes. The tetrahydrofluoride and dimethylformamide are distilled off and the residue is extracted with ethyl acetate containing a small amount of butanol. The extract was washed sequentially with 1N-citric acid, saturated aqueous sodium hydrogen carbonate solution, and saturated saline, and then with hot ethyl acetate to obtain Z-Pra-GJs-NH.
Obtain NHBac5-67f.

Rf, 0.60 1/, 0.76 Elemental analysis value (as C23H33N507) Calculated value (%
) C56,20H6,77N 14.25 Actual value (
11) C55,97H6,68#14. l52((
H-Pr#-Gln-mlNHB-to production Z
-Pro-G, mopngNHIJ, c 5.50 t
is dissolved in 50 dK of methanol and subjected to catalytic reduction using palladium as a catalyst to obtain H-pre-αmuNHNHB.

Rf”: 0.09 2(A), Z-Law-Pre-GLm-NIN-
The production of Z-Lag-0NH54-05f was described above (→
Get the fig-pr.

-01a -NHNH&-cC) "; Add to 100m of methyl fluoride and 1.56117 solution of triethyl acetate and leave at room temperature for 20 hours. After distilling off the dimethyl fluoride, the residue is extracted with ethyl acetate. The extract was washed successively with IN-citric acid and saturated saline.Reprecipitated with ethyl acetate-ether to give Z-L-buty-pr.-G.
L s -NHNIl&-c 3-72 Obtain f・R
f, 0.68 R7,0,80 Elemental analysis value (as 029H,4N6Q) Calculated value C'
S) C57,60H7,33N13.90 actual value (
1G) C57,21H7,08ff13.583(
g), H-Lgs-pre-GJs-NHNHB
KC) Manufacturing Z-Lag-Pro-GZs-NHNEEL
Dissolve t3.50 t in methanol 5Qsl, para!
;H-La by catalytic reduction using U as a catalyst
a -01s -NHNHBt is obtained.

Rf, o, s+ 3(h), Z-Asp(OCH3-C6H5')
-Lag-Pra- to Zs -NHNHBtse production Z-Asp(OCH2-C6HJ-OH2,27t t
TE) 5 Dissolve in 0.65 sl of Cyto0F5Si3Q+wj and N-methyl morpholin, and after cooling to -15°C, add 900 bottles of Isodunal and 0.84 ml of N-Metal morpholin and stir vigorously for 30 seconds. Nine H-Lag- obtained by above (-) on the back
dimethyl bookluma of prO-Cds-NHNHBac! Add 20ml of triethylamine solution and stir for 1 minute. After warming at 0°C for 5 minutes and then at 40°C for 2 minutes, the mixture is stirred at room temperature for 15 minutes. The tetrahydrofuran and dimethylformamide are distilled off, and the residue is extracted with ethyl acetate. The extract is washed successively with IN-citric acid, a saturated aqueous sodium hydrogen oxide solution, and saturated brine, and the extract is distilled off. Reprecipitate with ethyl acetate-ether-petroleum ether to give Z-,hp(OCH2-C6H,)
-Las-Pry -Ghb-NHNHIhc 4.
Get 00t.

Rf": 0.6g Rf, 0.77 Elemental analysis value (as C40'55N7'l+) Calculated value (
li) C59,32H6,84N12.11 Actual value ('3G) C58,88H6,65N 11.72
4(a), H-Asp-Law-1hr*-GLm
-Production of NHNII&x Z-Asp(OCH3-C6H
5) -Law, -pre-Of -NHNHIhc2.
00f was dissolved in methanol 50- and SO1 acetic acid 10 d, and catalytic reduction was performed using Ba5dium black as a catalyst to obtain H-Asp.
-Law-pro-Glh-NHNE13oc is obtained.

Rf, 0.08 4(b), Z-5ay-Asp-Law-pre
-Production of Glh-NHNHBocZ-5ay-NHNHa 0.75 f tdimethylformamide 15- and 6N-hydrochloric acid/geo-beef marbling 1.48
After dissolving in 1 Ll and cooling to -15°C, 0.39% of isoamyl nitrite was added and stirred for 5 minutes. Then, 1.24 mm of triethylamine is added for neutralization. Obtained by (α) above, H
-Asp-LaW-pry -GLyh-NHNHk dimethyl trichloride 1Qsu and triethylanine 0
．． Add the above reaction solution to the 341117m solution and incubate at 4℃ for 2 hours.
Stir for 0 hours. Dimethylformanide is distilled off and the residue is extracted with butanol. The extract is washed with water and the butanol is distilled off. Recrystallize from methanol-ethyl acetate,
Z -5ay-Asp -Law -pro -GLh
-NHNHBecl, 52F is obtained.

Rf”: 0.45 Rf, 0.67 5, H-5ty-Amp-Law-pl-a-G
Zn-Thr-His-5ar-LaM-Glh-Az
n-Arg-Arc-ALa-LaM-ILg-L
awr-Law-ALa-Glh-OHO production Z-5a
y-, 4#F-Law-pre-GZs-NHNHB
oe 11689 was removed with TFA2xt, anhydrous ether was added, and the resulting precipitate was dried with dimethyl ether. 5d and 6N-hydrochloric acid/geo beef sashimi 0.0?
21111j and cooled to -15°C. Add 0.019 d of triethyl anine and stir for 5 minutes. Then add 0.081 su of triethylanine to neutralize.

Peptide B or H-Tkr-His-5at-Lgs-
Ly-Ag-Arg-Ag-Alt-Las'-
11e-LaM-1ay-ALts-GZ%-0H8
The above reaction solution was added to a solution of 5 ml of Dimethyl Honrumuaed and 6 d of hesamethyl lysic acid triamide, and the mixture was stirred at 4° C. for 20 hours. Furthermore, Z −5ay−, 4zp −
Add 1164, an azide of 1ay-Pro-GLn-NHNHBoc, and stir for 28 hours. Dimethyl book rumua! The residue was extracted with ethanol. The extract is washed with water, butanol is distilled off, petroleum ether is added to the residue to crystallize it, the precipitated crystals are separated from P, and reprecipitated with methanol-ethyl acetate.

The obtained Z-5ar-Asp-Law-pro-GZ
+s -Tkr -Hlp-5at-Lass-Gly
-Azn-Arg-lrg-ALa-Last -I
Dissolve La-Law-Law, -jLet-Glh-OH in methanol 3Qsd and 10-acetic acid: 50117,
58 cape lil-5ar-noz after contact with palladium
p-Law-pry-(Ja-Thr-Hst-5ay
-Law, -GLy-jzs-Ag-Ivy-ALtx
-Law-ILe -Lag-Law -ALa-
Obtain s-ON. Hereinafter, this will be referred to as "Bezuchido C".

Rf": 0.01 Rf": 0.37 Elemental analysis value (C, 5I, 3N3 engineering '29, 2CH3C
As αW・7H20) Calculated value (-) C48,54f7.61 #17.
72 Actual value (III) 048.14 N7.50
N17.48[g]'7: -85,70<c-o
, 2s, 1M acetic acid) Production Example 4 1(''), H-5ay-ru p-Law-pre-GL%
-Production of NHNHB6c Z-5ay-lap-Lag-pre-Gln-NHN
Dissolve Hf3oc1.03 t in methanol 5Qm,
Catalytic reduction using palladium as a catalyst to produce H-5tr-A
OR/′ to obtain sp-LaK-pro-Gin-NHNElbc: 0.06·1(h), Z-Tyr-5ay-Asp-Lag-P
Production of ro-Gin-NHNHB6c Z-Tyr-ONH50,79f was prepared from the above (g)? Obtained H-5a r -Atp -L g * -Pr
o -Gl a -NHNHBoc (DdijfL Added to 20 iu solution of pyramide and left at room temperature for 20 hours. After distilling off the dimethyl rumamide, WI4fIi was extracted with ethyl acetate. The extract was mixed with IN-quesic acid and saturated brine. Wash sequentially with methanol and distill off ethyl acetate.
Reprecipitated with ethyl acetate to give Z-Tyr-5ay-Asp-
Lag-pre-GZs-NHNHlk 58 B
“I get l.

Rf": 0.51 Rf": 0.69 Elemental analysis value (as '52'69N9') Calculated value (
h) C58,91H6,56#11.89 actual measurement value (
%) C5B, 53 H6, 22N12.282,
H-Tyv-5ay-Asp-Law-Pry-
GJ%-Thr-HisSty-Lag-GLy-As
s-Arl-Arc-Ale-Law-11a-La
Production of m-1H irises-ALa-GLn-OK 2-Tyr-
5ay -lap-LaK -pry -GLn-NH
NHBoc4 4 cape dimethylformanid 3- and 6
Dissolved in N-hydrochloric acid / sashimi 0.0225111 /
Isoal nitrite after cooling to -15°C, 0060 I
Add IL/ and stir for 5 minutes. Then, 0.02521/ml of triethylanisi was added to neutralize.

Peptide B, namely H-TAr-Hip-5ay-1, an
-Gly -Ass-Arl-Arc-ALa-Lag
-ILe -Law-Law-Ala -GLn-OH
The above reaction solution was added to a solution of Dimetyllumamide 57 and Hesamethyllisic acid triamide 2117 of 25'I, and the mixture was stirred at 4°C for 20 hours. Furthermore, Z-Tyr
-Sur -Asp -Law -pra -Gin
- Add azide of NHNHB-C44”I,
Stir for 24 hours. The solvent was completely distilled off, the residue was extracted with butanol-water, added to an ether tube, and the precipitated crystals were collected at the bottom of the furnace.

Obtained 9Z-Tyr-5ay-Asp-Lag-pr
o-Gin-7'Ar-Hls -Say-Lgu-G
Ly-Ass-Arg-Arg-ALa-1ga
- Melt and catalytically reduce using rose rum as a catalyst.

The catalyst was removed and distilled off in a methanol tube, and the resulting residue was purified with Sephadex G-25 (eluent: 50 thiacetic acid), followed by LH-
20<elution tlL/,. H-T
yr-5ay-Asp-Lets-pry-Gin
-Thr -#l#-5ay-La@-GLy-Ass
-Arc -Arg-ALa-LaWL-ALa-L-
Magazine-LaK-ALa-GLn-OH18 cape is obtained. Hereinafter, this will be referred to as "Bepuyodo D".

Rf": 0.01 Rf": 0.38 Elemental analysis value (C engineering. 4H□69N32'3□・2 CH
As 3CO0H・5II20) Calculated value (1) C50,40H7,32N17.41
Actual value (11) C50, 72H7, 67N17.0
325.

[α), -77,88 (C-0,22,1M acetic acid) <Production of antigen> Production example 1 Synthesis of peptide. 15q of “BSA”)
acetic acid buffer (0.1 mol, pH 7, o)
2- Dissolve. To this solution is added 0.11 m/m of a 0.1 molar cultaraldehyde solution, and the mixture is stirred at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against 1 t of water. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is lyophilized to obtain trimmed plastoid interferon anti-M (hereinafter referred to as "antigen!") 18w.

This antigen (2) is a good product with peptide A bonding to BSAIV nil on average by 12 V nyl.Production Example 2 Peptide synthesis
20111 of A was dissolved in acetic acid a:J'F:Nium buffer (0,
17L, pH 7, 0>2-. 0.1 in this solution
Add 0.11 d of Kurtal aldide solution,
Stir at room temperature for 5 hours. The reaction mixture was then heated for 48 hours.
Dialyze against water It at 4°C. Change the water 5 times during dialysis. Thereafter, the solution containing the beta-protein complex was lyophilized to obtain trimmed plastidoid protein anti-J [(hereinafter referred to as "antigen") 23q.

The obtained antigen (2) has peptide B bound to ESJ11'el at an average of 9°C.

Production Example 3 Bedutide Synthesis 4.5 cape of Betutide C obtained in Production Example 3 and 2511F of ESA were added to ammonium acetate buffer (0, I
2d, pH 7,0'). O in this solution
, tV Nil O gel taraldehyde solution (1.0 d) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against 1 t of water. Change the water 5 times during dialysis.

Thereafter, the solution containing the bezutide protein complex is freeze-dried to obtain human rimphoblastoid titer-7e0-i (hereinafter referred to as "antigen 2") 27119.

The resulting nine antigens (1) are those in which peptide C is bound to B5A17 on average at 10°C.

Production Example 4 Bedutide synthesis 5q and BS of 9 Bepde D obtained in Production Example 4
Add 25 sv of A to acetic acid buffer solution (0.1 tjL).
,, pH 7,0') Dissolve in 2W'l. In this solution, Q,
Add 0.111+7 liters of cultaraldehyde solution and stir at room temperature for 5 hours. Then the reaction mixture was
Dialyze against 1 t of water at 4°C for hr. Change the water 5 times during dialysis. Thereafter, the solution containing the Bezutido protein complex is lyophilized to obtain human limhoplastoid isitaphae O antigen (hereinafter referred to as "antigen ■") 28m19.

The nine antigens obtained were
are bonded at an average of 9°C.

Production Example 5 4.5 capes of Bezuyodo C obtained in Production Example 3 and 25q of B5A are dissolved in 41 Lt of water. To this solution, 200 wf of disil-9-bocyinide (DCC) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture is then dialyzed against water 2 at 4° C. for 48 hours. Change the water 5 times during dialysis. Thereafter, the solution containing the beta-protein complex was lyophilized to obtain the Trim-su-5-stoid interferon antigen (hereinafter referred to as "antigen ■") 27.Ssv.

The obtained antigen V has an average of 12 molecules of peptide C bound to B511 molecules.

Production Example 6 Peptide Synthesis 4.5 capes of Bezutide D and B5A25'Q obtained in Production Example 4 were dissolved in 1jK of water. Add to this solution; Add 200q of DCC and stir at room temperature for 5 hours. The reaction mixture is then dialyzed against water 2 at 4° C. for 48 hours. Change the water 5 times during dialysis. The solution containing the protein complex is lyophilized to obtain the Trim Honzu Rustoid Interferon Antigen (hereinafter referred to as "Antigen ■") 29q.

The nine antigens obtained were peptide D against BSA1v.
has an average of 9r-nyl bonds.

<Antibody O production> Production example 1 100μf of antigen Io obtained in antigen production example 1 was added to 1.5 m□
After dissolving in physiological saline, add 1.5 s of Furuiside auxiliary solution.
A suspension prepared by adding / was added to three rabbits (2.5-3.
0Kg) K is administered subcutaneously, and the same amount is administered 6 times every 2 weeks. Furthermore, the same amount as the first dose is administered three times every fil month. Seven days after the final administration, blood was collected from the test animals and centrifuged to collect antiserum.
).

Production Example 2 20 μf of antigen ■ obtained in Antigen Production Example 2 was added to 1.5XtO
Physiological saline VC! After dissolution, add Furuiside auxiliary solution 1-.
A suspension prepared by adding 5II7 was added to seven rabbits (2,5
~3. Orw), and the same amount is administered 6 times every 2 weeks. Then, every 1 month thereafter, administer 3 [d, the same amount as the initial dose. m#! After 7 days have elapsed from the administration site, blood is collected from the test animal and centrifuged to collect antiserum to obtain the human plasmid antibody of the present invention (hereinafter referred to as "antibody 2").

Production Example 3 Anti-gt) 20μV of antigen obtained in Production Example 3 was 1.5-d
After dissolving in O saline salt water, add 70% Indian auxiliary solution 1.5
Add mt and prepare a cloudy solution to 7 rabbits (2.5-3.
Off'), administered subcutaneously, 6 times every 2 weeks.

Administer the same amount. After that, administer the same amount as the first dose three times every month. Seven days after the final administration, blood was collected from the test animals, centrifuged, and antiserum was collected. obtain.

Production Example 4 100 μm 11.5 dc of antigen ■ obtained in Antigen Production Example 3
After dissolving in 111 saline solution, add 70% Indian auxiliary solution 1.5
A suspension prepared by adding 117 was added to five rabbits (2,5-
3,04), and the same amount is administered 6 times every 2 weeks. *Administer the same amount as the first dose 3 times every month after treatment. Seven days after the final administration, blood was collected from the test animals, centrifuged to collect antiserum, and the plastoid interferon antibody of the present invention (hereinafter referred to as "antibody ■") was collected.
).

Production Example 5 2θμf t-1,5- of the antigen ■ obtained in Antigen Production Example 4
After dissolving in physiological saline, Freund's supplement 111.
A suspension prepared by adding 5117 to 7 rabbits (2,
5-3. Of+), and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the first dose was administered three times every month, or blood was collected from the test animal after 7 days from the final administration, and the antiserum was collected by centrifugation. This plastoid citrate antibody (hereinafter referred to as “
(referred to as "Antibody V") Production Example 6 100 pf of antigen (2) obtained in Antigen Production Example 4 was diluted with 1.5-
After dissolving in physiological saline, add 70% Indian auxiliary solution 1.5
Add su and prepare a cloudy solution to 3 rabbits (2.5~
3.04) K is administered subcutaneously, and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the first dose was administered three times every month. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect the antiserum to obtain the plastoid interferon antibody of the present invention (hereinafter referred to as "antibody ■").

Production Example 7 Preparation Example of Antigen Using 20 μt of anti-1 [V obtained in Example 7], a citrimophlastoid antibody (hereinafter referred to as "antibody 1") was obtained in the same manner as in Production Example 3 above.

Production Example 8 Using 100 μf of the antigen obtained in Preparation of Antigen Example 5, a human trimester interferon antibody (hereinafter referred to as "a-body matrix") is obtained in the same manner as in 1i) 6m4 above.

Production Example 9 A plastoid interferon antibody (hereinafter referred to as "antibody hawk") is obtained in the same manner as in Production Example 5 above using the anti-111c obtained in Antigen AM Preparation Example 6 > 20 μft.

Production Example 10 Using 10,100 μft of the S9 antigen in S9 Antigen Example 6, 5 rabbits were immunized in the same manner as in Production Example 4 above, and blood was collected from the test animals and the antiserum was collected by centrifugation. ,
EXAMPLE 1 Obtain the lactoid interferon antibodies (hereinafter referred to as "antibodies I and J") of the present invention.

0-labeled bezuide O production H-Tyr-5ay-Asp-Law-pry-GLm
-Thr-Hip -5ar-Lag-GZy-AsK
-Arc-Arc-ALg-4gg-11g -Lag
-LaK-jZa -GJa-OH, ie, Pezoid D, is underlabeled by the following method using Re05MishT.

That is, the above-mentioned bezuide 5μf O, 0.5f phosphate buffer (pff7.0) 20μtycyε (1) (eQr
rigr from N, LH,) Add 1 micro-medium Ury's 0.5 molar phosphate buffer, then chlorine 5:
l-shi T70 cape/d 0.5℃ luphosphate buffer 20μ
Add t. 0.5M of sodium metabisulfite (Ha2S205) at 60w5t/d with stirring for 30 seconds at room temperature.
The reaction is terminated by adding 50 μt of phosphate buffer. Next, 1. - cold sodium iodide aqueous solution 1
00 μt and the reaction mixture was transferred to Sephadex G-25.
C) Apply to column (1,Ox'J03) (eluent 0.25-BSl, 10M EDTA and 0.02
0.05 molar phosphate buffer containing ChN@Ns, pH
7,4'). The 13th and 14th fractions are marked with ① and are circled above.

Measurement of 0 titer The titer of the antibody obtained above is measured over the following days.

That is, the antibodies were dissolved in physiological saline at 1O1102,103, respectively.
, lO'', 105...fold dilution (initial),
To 100 μt of each of these, (1 ml and 0.05 v of labeled peptide (the labeled peptide obtained above was diluted to about 95,006 pN): Lusonate buffer (C pH 7.4)) ( 0,25chi ESA, I
Contains OmMEDTA and 0.0251 MILK3)
Add 0.2a/, incubate at 4°C for 24 hours,
The resulting conjugate of antibody and I-labeled antigen was subjected to De+Strane activity longitudinal method and centrifugation (4°C, 30 minutes, 300°C).
0rpm) K, unreacted (binding L) 1125ta is separated from the compound, its radiation is counted, and the binding rate (-) of the antibody with the I-labeled peptide at each dilution concentration is determined.
Measure. The vertical axis represents the binding rate (s) of the antibody with the 1125 standard fiber betaid, and the horizontal axis represents the dilution rate (initial concentration) of the antibody, and the binding rate is lofted at each concentration.

The dilution ratio of the antibody at which the binding rate is 50 degrees, that is, the antibody titer is determined. The results are shown in Table 1 below.

Table 1: As a specificity test sample for human plastid Isiter feuer of O antibody, each concentration of β interfere (
Manufactured by Tokyo Metropolitan Clinical Research Institute, specific activity 3 x 106 U/III
Protein), Peptide C obtained in VeptiFO Synthesis Production Example 3, VeptiF- and Shil-4JSll Isiturf Ion [manufactured by Zainsei Research Institute, Rimhon Plastoid Inter] Hue 0shi Let,
) h, 800928 and Kashityl (C axis tag company 11)
]. In addition, 0.25% BSA is used as a standard diluent.
, 0.05'E luricate laxative solution (s47-4) containing 50M EDTA and 0.029 ION gps is used.

In each test tube, add 0.1 d of the standard diluent, 0.1 d of the sample sample, 0.1 d of the antibody (200,000 titer) obtained in the antibody production example, and I-labeled Bezuide (as described above). The obtained labeled peptide was diluted to about 2800 epwm, added with 0.1-0.1 epwm of epwm, and incubated at 4°C for 72 hours.
Add 0.1 m of German (eing german), then coat with deboss straw and add 0.5 m of a suspension of activated charcoal.
Let stand at 4°C for 50 minutes, then centrifuge at 4°C for 50 minutes at 5o00 rpm to remove the antibody and 112
Conjugate with 5-labeled bezuide and unreacted (does not bind)■
Separate the labeled bezuide, remove its radiation,
The binding rate (Bo ) corresponding to the titer of the antibody used is loo
As 'l', determine the percentage of the conjugate (B) between the antibody and the target bezuide at the concentration and dilution rate of each test sample. The results obtained are shown in FIG. The vertical axis in Figure 1 is the connection *
(B/Bo It shows the concentration of c). Also, in the diagram, curve (a) represents Bezuid C, that is, the Bezuid chain of human plastoid interferon. !
(b) is human α-type interferon 0 (manufactured by Kancil), music is<c) is human a-type interfacial interferon 0 (written by the Materials Research Institute), and 9-line (d) is Antibodies (Fig. 1) show a group of antibodies that are clearly differentiated in their reactivity to α-inhibitory interference and to β-inhibitory interference, and from this. 〉What is β-type interferon 0:I?3. OX
It can be seen that this is a highly specific antibody that does not cross to 10'l nits/-.

The antigens I to m obtained in the above anti-IFO production examples 1 to 6
The binding rate of VeptiF and ll5A in K is determined for each antigen 7I! Kt FADAX G-50CILIj liquid:
Raw hot water food woodcutter, detection: OD 289 m Wei, outflow 11 J
I: 3 m/hour, aliquoting amount: 1 g each), and no unreacted 851 or VeptiFID was observed).
The futakushiyoshi of the peptide dimer and the futakushiyoshi of another product (VeptiFO dimer) were separated, and the standard source of the peptide dimer, Jlf, was separated.
A calibration curve was created to determine the amount of the dimer, and this was subtracted from the amount of VeptiFO used as the starting material, and the value was calculated assuming that all of the value was bound to BSA.

[Brief explanation of the drawing]

FIG. 1 shows a club showing the characteristics of the trimmed plastic toy F pile body obtained by the present invention. (or more) ->

Claims (1)

[Claims] ■ General formula % Formula % [The hydrogen atom in the formula is H-Thv-Hip-5ay-L
aw-(dy-Ass-Arg-Arg-Arg group, H
-5edayhp-Lag-Pr#-GZs-The-H
ip-5ay-Lg@-GLy-Ass-Arg-A
rg-Arg group, or H-Tyr-5at -Asp-
J, gs-Pro-GZs-Thr-Hip-5a
y-Lax-GLy-Ass-Arc-Arg-11
Indicates the g group. ] A heptomeplastoid isiter antigen consisting of a heptomeplastoid IsiterF 0SON-terminal peptide and at least one of its O# conductors, a carrier, and an O complex is transferred to a mammalian body. 1. A method for producing an antibody, which comprises administering the antibody to a patient and harvesting the produced antibody.